CN110650745A - Armed replicative oncolytic adenoviruses - Google Patents

Abstract

Replication competent oncolytic adenoviruses comprising a chimeric human/mouse CD40 ligand are disclosed. The oncolytic adenovirus may be replicative. The chimeric human/mouse CD40 ligand can be MEM 40. Also disclosed are methods comprising administering to a patient having cancer a replicative oncolytic adenovirus having at least one chimeric human/mouse CD40 ligand (e.g., MEM 40).

Description

Armed replicative oncolytic adenoviruses

Background

This application claims priority to U.S. provisional application 62/437,474 filed on 21/12/2016 and U.S. provisional application 62/584,008 filed on 9/11/2017, both of which are incorporated herein by reference.

Technical Field

The present invention relates generally to the fields of virology, immunology and medicine. More particularly, it relates to compositions of oncolytic adenoviral vectors for the treatment of cancer.

Background

Oncolytic viruses are a class of cancer therapeutics with a dual mechanism of action: 1) killing the tumor cells by selective viral replication in the tumor cells, resulting in direct tumor lysis; 2) systemic anti-tumor immunity is induced by the release of antigens in the destroyed tumor cells. Both native and transgenic viruses are under development. The first oncolytic virus, taliomogene laherparepvec, (U.S. FDA approved in 2015

Amgen inc., thunusand Oaks, CA), a transgenic herpesvirus encoding granulocyte-macrophage colony stimulating factor (GM-CSF) for use in the local treatment of melanoma, as described by Kohlhapp et al.2016clinical Cancer Research. However, melanoma is only one of many cancers. In addition, GM-CSF is only one of many compounds under investigation for cancer therapy. In addition, herpes virus is only one of many viruses whose oncolytic properties have been implicated in its research.

Thus, there remains a need for oncolytic viral vectors for the treatment of cancer.

All subject matter discussed in the background section is not necessarily prior art and should not be admitted to be prior art merely by virtue of its discussion in the background section. In addition, identification of any problems with the prior art discussed in the background section or related to these subject matter should not be considered prior art unless explicitly stated as prior art. Rather, the discussion of any subject matter in the background section should be considered part of the inventor's approach to a particular problem, which may itself be inventive.

Disclosure of Invention

The following presents a simplified summary of the disclosure in order to provide a basic understanding of some aspects of the disclosure. This summary is not an extensive overview of the disclosure. It is not intended to identify key or critical elements of the disclosure or to delineate the scope of the disclosure. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is discussed later.

In some embodiments, the disclosure relates to oncolytic adenoviruses comprising a chimeric human/mouse CD40 ligand. The oncolytic adenovirus may be replicative.

In some embodiments, the present disclosure relates to a method comprising administering to a patient having a tumor a composition comprising an oncolytic adenovirus comprising at least one chimeric human/mouse CD40 ligand.

In any embodiment, the chimeric human/mouse CD40 ligand can be selected from ISF30-ISF41, wherein MEM40(ISF35) is a member thereof.

Transgenes encoding chimeric human/mouse CD40 ligands selected from ISF30-ISF41, including MEM40(ISF35), have previously been inserted into non-oncolytic, non-replicative adenoviruses. However, these transgenes have never been designed as oncolytic and/or replicative adenoviruses.

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter, unless explicitly stated otherwise.

The details of one or more embodiments are set forth in the description below. Features illustrated or described in connection with one exemplary embodiment may be combined with features of other embodiments. Thus, any of the various embodiments described herein can be combined to provide further embodiments. Aspects of the embodiments can be modified, if necessary, to employ concepts of the various patents, applications and publications identified herein to provide yet further embodiments. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

Drawings

The disclosure may be understood by reference to the following description taken in conjunction with the accompanying drawings, in which:

FIG. 1 schematically shows a construct of a novel replication competent adenovirus Delta-24-RGD-MEM40(DNX-MEM40) expressing MEM 40.

FIG. 2 schematically shows a construct of a novel replication competent adenovirus Delta-24-MEM40 expressing MEM 40.

Detailed Description

Various illustrative embodiments of the disclosure are described below. In the interest of clarity, not all features of an actual implementation are described in this specification. It will of course be appreciated that in the development of any such actual embodiment, numerous implementation-specific decisions must be made to achieve the developers' specific goals, such as compliance with system-related and business-related constraints, which will vary from one implementation to another. Moreover, it will be appreciated that such a development effort might be complex and time-consuming, but would nevertheless be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure.

The present subject matter will now be described with reference to the drawings. Various structures are schematically depicted in the drawings for purposes of explanation only and so as to not obscure the present disclosure with details that are well known to those skilled in the art. However, the attached drawings are included to describe and explain illustrative examples of the present disclosure. The words and phrases used herein should be understood and interpreted to have a meaning consistent with the understanding of those words and phrases by those skilled in the relevant art. Special definitions for terms or phrases, i.e., definitions that differ from the ordinary and customary meaning as understood by those skilled in the art, are intended to be implied by consistent usage of the term or phrase herein. To the extent that a term or phrase is intended to have a special meaning, i.e., a meaning other than that understood by skilled artisans, such a special definition will be expressly set forth in the specification in a definitional manner that directly and unequivocally provides the special definition for the term or phrase.

While the subject matter disclosed herein is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described in detail. It should be understood, however, that the description herein of specific embodiments is not intended to limit the disclosure to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the disclosure as defined by the appended claims.

Overview of cancer

"cancer" refers to a large family of diseases characterized by uncontrolled growth of cells in vivo. Representative forms of cancer include carcinomas, sarcomas, myelomas, leukemias, lymphomas, and mixed types of the foregoing. Further examples include, but are not limited to, those discussed in more detail below.

Overview of adenovirus

"adenovirus" (Ad) is a large (about 36kb) DNA virus that infects humans, but also exhibits a broad host range. Physically, an adenovirus is an icosahedral virus that contains a double-stranded linear DNA genome. There are approximately 50 human adenovirus serotypes, which are classified into 6 families according to molecular, immunological and functional criteria. By adulthood, almost everyone has become infected with the more common adenovirus serotype, the primary effect being cold-like symptoms.

Adenoviral infection of the host cell results in episomal maintenance of the adenoviral DNA, which reduces the potential genotoxicity associated with the integrating vector. Furthermore, adenoviruses are structurally stable and no genomic rearrangements were detected after extensive amplification. Adenovirus infects most epithelial cells, regardless of the cell cycle phase. To date, adenovirus infection appears to be associated with only mild diseases, such as acute respiratory disease in humans.

The infection cycle of adenovirus is carried out in two steps: it allows the production of regulatory proteins and proteins involved in viral DNA replication and transcription, at an early stage prior to initiation of adenovirus genome replication, and a late stage leading to structural protein synthesis. Early genes are distributed in 4 regions scattered in the adenovirus genome, designated as E1 to E4 ("E" means "early"). The early region contains at least six transcriptional units, each with its own promoter. The expression of early genes is itself regulated, with some genes being expressed before others. The three regions E1, E2 and E4 are essential for virus replication. Thus, if an adenovirus is defective for one of these functions, the protein must be provided in trans, or the virus cannot replicate.

The E1 early region is located at the 5' end of the adenovirus genome and contains 2 viral transcription units E1A and E1B. This region encodes a protein involved very early in the viral cycle and is essential for the expression of almost all other genes of the adenovirus. In particular, the E1A transcriptional unit encodes a protein that transactivates transcription of other viral genes, inducing transcription of the E1B, E2A, E2B, E3 and E4 regions and late genes from the promoter.

Adenoviruses enter permissive host cells via cell surface receptors and are then internalized. Viral DNA associated with certain viral proteins required for the first step of the replication cycle enters the nucleus of the infected cell where transcription begins. Replication of adenoviral DNA occurs in the nucleus of infected cells, and cell replication is not required. New virus particles or virions are assembled, after which they are released from the infected cells and can infect other permissive cells.

Adenovirus is an attractive delivery system. Embodiments of the present disclosure may utilize fabrication methods with maximum yield of 1 × 10 per cell⁵And (c) viral particles. The method can be free or substantially free of proteins, serum, and animal-derived components, making it suitable for use in a wide range of prophylactic and therapeutic vaccine products.

If the adenovirus has been mutated so that it is conditionally replicative (replication competent under certain conditions), helper cells may be required for viral replication. Helper cell lines can be derived from human cells, such as human embryonic kidney cells, muscle cells, hematopoietic cells, or other human embryonic mesenchymal or epithelial cells, as desired. Alternatively, the helper cells may be derived from cells of other mammalian species that are permissive for human adenovirus. Such cells include, for example, Vero cells or other monkey embryonic mesenchymal or epithelial cells. In certain aspects, the helper cell line is 293. Various methods of culturing host cells and helper cells can be found in the art, for example (Racher, A.J., Fooks, A.R. & Griffiths, J.B.Biotechnol Tech (1995)9: 169.).

Different methods can be used to isolate adenoviruses. Most commonly, after transfection of the Ad genome, adenovirus plaques are isolated from agarose-covered cells and the virus particles are expanded for analysis. For detailed protocols, reference is made by the person skilled in the art (Graham, F.L., and Prevec, L. (1991). Manipulation of adonovirusvectors. methods Mol Biol 7, 109-.

Alternative techniques for generating adenoviral vectors include the use of Bacterial Artificial Chromosome (BAC) systems, in vivo bacterial recombination of recA + bacterial strains using two plasmids containing complementary adenoviral sequences, and Yeast Artificial Chromosome (YAC) systems (PCT publications 95/27071 and 96/33280, incorporated herein by reference).

Representative examples of adenoviral vectors suitable for use in the present disclosure include those described in U.S. publication nos. 2009/0175830, 2014/0377221, 2014/0377294, 2015/0306160, 2016/0289645 and 2016/0143967 and U.S. patent nos. 6,210,946, 6,284,742, 6,312,699, 6,555,368, 6,649,396, 6,815,200, 6,824,771, 6,841,540, 6,955,808, 7,045,348, 7,297,542, 8,168,168 and 9,061,055, all of which are incorporated herein by reference in their entirety.

Overview of oncolytic viruses

A wide range of oncolytic virus types are being developed as anti-cancer agents, including adenoviruses (see Russell et al, 2014Nature Biotechnology and Lawler et al, 2017JAMA Oncology).

A variety of biological properties may be considered in selecting or designing a therapeutic oncolytic adenovirus for a desired therapeutic activity, including: selectively targeting cancer cells for infection by natural tendency of cell surface proteins or by direct targeting of engineered adenoviruses to cancer cells; selective replication in cancer cells; the pathogenesis of the virus is weakened; enhancing the dissolution activity; modifying the antiviral immune response, which can result in rapid clearance of adenovirus; and modifying the systemic anti-tumor immunity by genetic modification of the adenovirus to incorporate a cytokine, an immune agonist, or an immune checkpoint blocker.

Replicative oncolytic adenoviral vectors have a variety of properties that make them ideal candidates for therapeutic applications, including infectivity of a wide range of cell and tumor types, infection of non-dividing cells, lack of genomic incorporation, high titer, ability to carry transgenes, in vitro and in vivo stability, and high level expression of transgenes. Adenoviral expression vectors include constructs comprising adenoviral sequences sufficient to (a) support packaging of the construct and (b) ultimately express the recombinant gene construct that has been cloned therein.

Modulating the biological properties of oncolytic adenoviruses can affect a range of immune interactions that may be beneficial or detrimental to cancer therapy. The interaction depends on the particular tumor, the location and extent of the disease, the immunosuppressive tumor microenvironment, the Oncolytic viral platform, dose, time and delivery conditions, as well as the individual patient response (see generally Aurelian L. "Oncolytic viruses as immunological therapy: progress and remainingchannels" one. targets Ther.2016; 9: 2627-. For example, it has been reported that The presence of The adenovirus E3 gene increases The in vitro and in vivo oncolytic potency of conditionally replicating adenoviruses (see Suzuki K, Alemany R, Yamamoto M, and Curiel DT "The presence of The adenoviral E3 regions improvements The oncolytic sites of regulatory replication ` Clin. cancer Res.2002Nov; 8(11): 3348-59). Specifically, E3-11.6kDa Adenovirus Death Protein (ADP) was thought to be essential for efficient cell death (see Tollefson A, Ryerse J, and Scaria A, et al, "The E3-11.6kDa Adenoviral Death Protein (ADP) is required for efficient cell death: chromatography of cells infected with ADP molecules," Virology 1996; 220: 152-. However, for immunotherapeutic approaches to the treatment of cancer, it may be important to balance rapid cell death with adequate expression of immunomodulatory proteins to optimally induce an anti-cancer immune response. The present disclosure provides such oncolytic adenoviruses.

According to the present disclosure, members of any of the 57 human adenovirus serotypes (HAdV-1 to 57) may incorporate heterologous nucleic acids encoding immune cell stimulating receptor agonists. Human Ad5 is well characterized genetically and biochemically (GenBank M73260; AC 000008). Thus, in particular embodiments, the oncolytic adenovirus is a replicative Ad5 serotype or a hybrid serotype comprising an Ad5 component. The adenovirus may be a wild-type strain, but may be genetically modified to enhance tumor selectivity, for example, by reducing the ability of the virus to replicate in normal quiescent cells without affecting the ability of the virus to replicate in tumor cells. Non-limiting examples of replication competent oncolytic adenoviruses encompassed by the present disclosure include Delta-24, Delta-24-RGD, ICOVIR-5, ICOVIR-7, ONYX-015, ColoAd1, H101, and AD 5/3-D24-GMCSF. Onyx-015 is a hybrid of viral serotypes Ad2 and Ad5, with deletions in the E1B-55K and E3B regions to enhance cancer selectivity. H101 is a modified version of Onyx-015. ICOVIR-5 and ICOVIR-7 contained a deletion of the Rb binding site of E1A and a substitution of the E1A promoter by the E2F promoter. ColoAd1 is a chimeric Add11p/Ad3 serotype. AD5/3-D24-GMCSF (CGTG-102) is a serotype 5/3 capsid-modified adenovirus encoding GM-CSF (Ad5 capsid protein knob (knob) is replaced by knob domain of serotype 3).

Oncolytic adenoviruses injected into tumors induce cell death and release new adenoviral progeny that generate therapeutic waves by infecting neighboring cells, which can lead to complete destruction of the tumor if not halted. The significant antitumor effects of Delta-24 have been shown in cell culture systems and in malignant glioma xenograft models. Delta-24-RGD has been shown to have anti-tumor effects in phase I clinical trials and is currently the subject of other clinical trials. Although tumor cell lysis is the primary anti-cancer mechanism of Delta-24-RGD oncolytic adenoviruses, data from phase I clinical trials of recurrent gliomas and other observed patients suggest that triggering of an anti-tumor immune response mediated by adenovirus can enhance direct oncolytic effects.

In some embodiments of the disclosure, one or more heterologous sequences may be incorporated into a non-essential region of an adenovirus. In a particular embodiment of the disclosure, one or more heterologous sequences may be incorporated in place of all or part of the E3 region. Representative examples include cytokines, chemokines, and checkpoint inhibitors. In some embodiments, the heterologous sequence encodes an OX40 agonist (e.g., OX40L), GITRL, anti-PD-1, and/or anti-CTLA-4. In another embodiment, the heterologous nucleic acid sequence encodes an inhibitor of an immune checkpoint protein selected from the group consisting of CTLA4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, TIM3, GAL9, LAG3, VISTA, KIR, and/or BTLA. In another embodiment, the heterologous nucleic acid sequence encodes an agonist of an immune co-stimulatory receptor selected from the group consisting of CD28, OX40(CD134), glucocorticoid-induced TNF receptor (GITR), CD137(4-1BB), herpes virus entry mediator a (hvem), inducible T cell co-stimulatory molecule (ICOS or CD278), CD27, CD40, and/or CD 226. Representative examples are disclosed in more detail in PCT/US2014/066920 filed on 27/5/2016 and in U.S. temporary number 62/342482, both of which are incorporated herein by reference in their entirety.

DNX-2401

In some embodiments, the replication competent oncolytic adenovirus is Delta-24 or Delta-24-RGD (DNX-2401). Delta-24 is described in U.S. patent application publication Nos. 20030138405 and 20060147420, each of which is incorporated herein by reference. Delta-24 adenovirus is derived from adenovirus type 5 (Ad-5) containing a 24 base pair deletion in the CR2 portion of the E1A gene, which comprises the region responsible for binding to the Rb protein (nucleotide 923-946) corresponding to amino acids 122-129 in the encoded E1A protein (Fueyo J et al, Oncogene, 19: 2-12 (2000)). Delta-24-RGD further comprises the RGD-4C sequence, which binds strongly to α v β 3 and α v β 5 integrins, inserted into the HI loop of the fibulin (Pasqualini R. et al, NatBiotechnol, 15: 542-546 (1997)). Deletion of E1A increases the selectivity of the virus for cancer cells; the RGD-4C sequence increases the infectivity of the virus to gliomas and several other tumors that express low levels of adenoviral receptors.

Summary of CD40 agonists

While not being bound by theory, the success of cancer immunotherapy may depend on enhancing tumor-specific CD8+ T cell immunity, as CD8+ T cells are intimately associated with direct tumor killing and patient survival. Therefore, therapeutic modalities that promote CD8+ T cell responses are the target of cancer immunotherapy drug development. The CD40 receptor is a member of the tumor necrosis factor receptor family and is expressed by B cells, professional antigen presenting cells and non-immune cells and tumors (van Kooten et al, 2000Journal of Leukocyte Biology). Activation of tumor-specific T cell responses requires activation of the CD40 receptor on antigen presenting cells. CD4+ T cells permit Dendritic Cells (DCs) to enhance CD8+ T cell priming through CD40-CD154 interactions. In addition, CD40-CD154 interaction may prevent premature reduction of CD8+ T cell responses. Thus, activation of CD40 by CD154 is an essential step in the induction of effective antigen-specific CD8T cellular immunity against pathogens and tumors (Singh et al, 2017Nature Communications). In this regard, CD 40-agonist therapy, such as antibodies or homologous CD40 ligand (CD40L) proteins, appears to be a promising strategy for cancer immunotherapy.

ISF35 overview

ISF35 (which may also be referred to herein as "MEM 40") is a chimeric human/mouse CD40 ligand that has 92% amino acid sequence homology to human CD 40L. See U.S. patent No. 7,495,090, which is incorporated herein by reference). ("CD 40 ligand" and "CD 40L" are used interchangeably herein and may also be referred to as "CD 154"). Specifically, domains I, II and III, the regions that comprise the intracellular, intramembranous and proximal extracellular domains of the molecule, respectively, have been fully humanized. In domain IV, which contains the CD 40-binding portion of the molecule, only those murine domains are retained that are necessary for optimal CD40 ligand expression in the cell. ISF35(MEM40) was fully humanized at the 3' end of the molecule, where antibody binding neutralizes the activity of murine CD154(CD40 ligand) when administered to humans. In addition to MEM40, there was a series of chimeric CD40 ligand constructs (ISF30 to ISF41), of which MEM40 is the specific chimeric CD40 ligand in this group, with the most preclinical and clinical experience.

Illustrative embodiments

In some embodiments, the present disclosure relates to oncolytic adenoviruses comprising at least one chimeric human/mouse CD40 ligand.

In some embodiments, the present disclosure relates to a replicative oncolytic adenovirus comprising at least one chimeric human/mouse CD40 ligand. In a particular embodiment, the replication competent oncolytic adenovirus may be genetically modified to incorporate a chimeric human/mouse CD40 ligand transgene.

In some embodiments, the present disclosure relates to a replication competent oncolytic adenovirus comprising a sequence encoding a CD40 agonist operably linked to a transcriptional control element.

In other embodiments, the disclosure relates to a replicative oncolytic adenovirus comprising a chimeric human/mouse CD40 ligand and one or more additional immunomodulatory or therapeutic genes, such as a cytokine (e.g., GM-CSF, TNF), an interleukin (e.g., IL-2, IL-12), a chemokine (e.g., RANTES), a macrophage inflammatory protein (e.g., MIP-3), a checkpoint inhibitor (e.g., anti-PD-1, anti-CDTA 4, and anti-PD-L1), or another immunomodulatory protein (e.g., OX40 ligand).

The genomic region of an oncolytic adenovirus can be altered for a variety of purposes to confer desired therapeutic properties. Non-limiting examples of therapeutic properties may include enhanced viral replication and spread, enhanced oncolytic effects, preferential targeting of tumor cells over normal cells, enhanced immune activation, and protection of adenovirus from the host immune system. The viral regions used for the above purposes may be eliminated (deleted in whole or in part), rendered non-functional, modified to reduce function, or substituted with other sequences.

In some embodiments, the present disclosure provides adenoviruses with improved tumor cell immune-mediated and viral destruction capabilities.

In some embodiments, the present disclosure relates to a method comprising administering to a patient having a tumor a composition comprising an oncolytic adenovirus comprising at least one chimeric human/mouse CD40 ligand.

Any oncolytic adenovirus strain can be used as a starting point for incorporation of the chimeric human/mouse CD40 ligand transgene. In some embodiments, the oncolytic adenovirus is a replicative human type 5 adenovirus.

Genetic modification of oncolytic adenoviruses to incorporate a chimeric human/mouse CD40 ligand transgene can be performed using techniques known to those of ordinary skill in the art. Oncolytic adenoviruses comprise a chimeric human/mouse CD40 ligand transgene (cytomegalovirus (CMV) promoter or alternative promoter; polyadenylation domain) with the necessary regulatory elements to allow transcription of the chimeric human/mouse CD40 ligand gene and expression of the chimeric human/mouse CD40 ligand polypeptide in infected cells.

In some embodiments, chimeric human/mouse CD40 ligand may be selected from the group consisting of ISF30(SEQ ID NO: 1), ISF31(SEQ ID NO: 2), ISF32(SEQ ID NO: 3), ISF33(SEQ ID NO: 4), ISF34(SEQ ID NO: 5), ISF35(MEM40) (SEQ ID NO: 6), ISF36(SEQ ID NO: 7), ISF37(SEQ ID NO: 8), ISF38(SEQ ID NO: 9), ISF39(SEQ ID NO: 10), ISF40(SEQ ID NO: 11), and ISF41(SEQ ID NO: 12).

Polynucleotide sequences encoding polypeptide sequences of ISF30, ISF32, ISF34, ISF36, ISF38, and ISF40 are disclosed in U.S. patent No. 7,928,213, which is incorporated herein by reference. Polynucleotide sequences encoding polypeptide sequences of ISF31, ISF33, ISF35, ISF37, ISF39, and ISF41 are disclosed in U.S. patent No. 7,495,090, which is incorporated herein by reference.

In particular embodiments, the chimeric human/mouse CD40 ligand incorporated into a replication competent oncolytic adenovirus can be ISF35(MEM40) (SEQ ID NO: 6).

The heterologous chimeric human/mouse CD40 ligand can be inserted into any nonessential location of the oncolytic adenovirus. In some embodiments, the oncolytic adenovirus remains replication competent. In particular embodiments, a heterologous chimeric human/mouse CD40 ligand nucleic acid is inserted into the E3region of the replicative adenovirus backbone. The E3region is not essential for viral replication. Replication competent adenoviruses may comprise a deletion of all or part of E3. In a related aspect, the entire E3region is deleted from the replicative adenovirus backbone and a chimeric human/mouse CD40 ligand nucleic acid is inserted into the position containing the entire E3 deletion.

The oncolytic adenovirus may be further genetically modified to improve one or more properties for treating cancer, including selective replication in cancer cells; the pathogenesis of the virus is weakened; enhancing the dissolution activity; modification of the antiviral immune response that results in rapid clearance of the adenovirus; and modification of virus-induced systemic anti-tumor immunity.

In particular embodiments, the present disclosure provides Delta-24 or Delta-24-RGD adenoviruses comprising an inserted chimeric human/mouse CD40 ligand nucleic acid in place of a partially or fully deleted E3region, wherein the heterologous nucleic acid comprises a sequence encoding a chimeric human/mouse CD40 ligand transgene.

In some embodiments, the disclosure relates to a method comprising administering to a patient having a tumor a composition comprising at least one chimeric human/mouse CD40 ligand comprising a replication competent oncolytic adenovirus. In particular embodiments, the oncolytic adenovirus and the at least one chimeric human/mouse CD40 ligand may be as described above.

In some embodiments, the oncolytic adenovirus comprising at least one chimeric human/mouse CD40 ligand can be a Delta-24-MEM40 replicative adenovirus.

In another embodiment, the oncolytic adenovirus comprising at least one chimeric human/mouse CD40 ligand can be a Delta-24-RGD-MEM40 replicative adenovirus.

Regardless of the choice of replicative oncolytic adenovirus and chimeric human/mouse CD40 ligand, a replicative oncolytic adenovirus comprising at least one chimeric human/mouse CD40 ligand may be used to treat cancer, wherein the adenovirus is administered by intratumoral injection. However, other routes of delivery are also contemplated, including intravenous, intraperitoneal, intratracheal, intramuscular, intracranial, endoscopic, intralesional, transdermal, subcutaneous, topical, or by direct injection or infusion.

While not being bound by theory, a replicative oncolytic adenovirus comprising at least one chimeric human/mouse CD40 ligand may have a dual mechanism of action: 1) killing of tumor cells by selective viral replication of oncolytic adenoviruses in cancer cells, and 2) induction of systemic anti-tumor immunity resulting from viral immunity induction and immune activation of CD40 ligand.

Adjusting element

Expression cassettes included in vectors useful in the present disclosure contain (in a5 'to 3' orientation) a transcription promoter operably linked to a protein coding sequence, a splicing signal including an insertion sequence, and a transcription termination/polyadenylation sequence. Promoters and enhancers, which control the transcription of protein-encoding genes in eukaryotic cells, are composed of a variety of genetic elements. The cellular machinery collects and integrates the regulatory information delivered by each element, allowing different genes to evolve different, often complex, transcriptional regulatory patterns. Promoters useful in the context of the present disclosure include constitutive, inducible, and tissue-specific promoters.

Promoters/enhancers

Chimeric human/mouse CD40 ligand nucleic acid expression may be under the control of a promoter functional in mammalian cells, preferably human tumor cells. Chimeric human/mouse CD40 ligand nucleic acid expression may be under the control of a non-adenoviral promoter. In one embodiment, the promoter directing expression of chimeric human/mouse CD40 ligand is the Cytomegalovirus (CMV) promoter.

The expression constructs provided herein comprise a promoter that drives expression of a programmed gene. Promoters generally comprise sequences that serve to locate the start site of RNA synthesis. The best example is the TATA box, but in some promoters lacking a TATA box, such as the promoter of the mammalian terminal deoxynucleotidyl transferase gene and the promoter of the SV40 late gene, discrete elements covering the start site themselves help to fix the start position. Additional promoter elements regulate the frequency of transcription initiation. These are typically located in the region 30 to 110bp upstream of the start site, although it has been shown that promoters also contain functional elements downstream of the start site. In order for a coding sequence to be "under the control of a promoter," the 5 'end of the transcription start site of the transcription reading frame is positioned "downstream" (i.e., 3') of the selected promoter. An "upstream" promoter stimulates transcription of DNA and promotes expression of the encoded RNA.

The spacing between promoter elements is typically flexible, thus preserving promoter function when the elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50bp before activity begins to decline. Depending on the promoter, it appears that the individual elements may act synergistically or independently to activate transcription. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

Promoters may be naturally associated with nucleic acid sequences and obtained by isolating 5' non-coding sequences located upstream of the coding segment and/or exon. Such promoters may be referred to as "endogenous". Similarly, an enhancer may be naturally associated with a nucleic acid sequence located downstream or upstream of the sequence. Alternatively, certain advantages are obtained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, i.e., a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other adenovirus or prokaryotic or eukaryotic cell, as well as promoters or enhancers that are not "naturally occurring," i.e., contain different elements of different transcriptional regulatory regions, and/or mutations that alter expression.

It may be important to use a promoter and/or enhancer that effectively directs the expression of a DNA fragment in the organelle, cell type, tissue, organ or organism selected for expression. One skilled in the art of molecular biology generally knows the use of promoters, enhancers and cell type combinations for protein expression. The promoters used may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA fragments, e.g., to facilitate large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

Non-limiting examples of promoters include early or late viral promoters, such as the SV40 early or late promoter, Cytomegalovirus (CMV) immediate early promoter, Rous Sarcoma Virus (RSV) early promoter; and eukaryotic promoters.

Initiation signal and Linked representation

Specific initiation signals may also be used in the expression constructs provided in the present disclosure for efficient translation of the coding sequence. These signals include the ATG initiation codon or adjacent sequences. It may be desirable to provide exogenous translational control signals, including the ATG initiation codon. One of ordinary skill in the art will be readily able to provide the necessary signals. It is well known that the initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translational control signals and initiation codons can be natural or synthetic. Expression efficiency may be enhanced by including appropriate transcriptional enhancer elements.

In certain embodiments, the use of an Internal Ribosome Entry Site (IRES) element is used to generate multigene or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylated cap dependent translation and begin translation at internal sites (Pelletier et al, 1988Molecular and Cellular Biology). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier et al, 1988Molecular and Cellular Biology), as well as IRES from mammalian information (Macejak et al, 1991 Nature). The IRES element may be linked to a heterologous open reading frame. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. The IRES element allows ribosomes access to each open reading frame for efficient translation. Multiple genes can be efficiently expressed to transcribe a single message using a single promoter/enhancer (see U.S. Pat. nos. 5,925,565 and 5,935,819, each incorporated herein by reference).

Virus modification method

The various genes mentioned may be functionally inactivated by several techniques well known in the art, including gene deletion, substitution or insertion. Similarly, immune modulatory genes, including MEM40, can be inserted into the viral genome by methods well known to those skilled in the art. These types of modifications in adenoviruses can be made by homologous recombination methods. For example, adenoviral genomic DNA can be transfected with a plasmid vector containing a mutant sequence flanked by homologous adenoviral sequences, resulting in DNA recombination and replacement of a region of the parental adenoviral genomic DNA with a new mutant sequence region.

Exemplary constructs of replicative oncolytic adenoviruses comprising chimeric human/mouse CD40 ligands are shown in FIGS. 1-2.

FIG. 1 schematically shows a construct of Delta-24-RGD-MEM40(DNX-MEM40), a replicative adenovirus expressing MEM 40. DNX-MEM40 is shown. Briefly, the 24 nucleotide sequence was deleted from the E1 region. Further, the E3 area is deleted. The 27 nucleotide sequence encoding RGD peptide CDCRGDCFC was also inserted into the H1 knob domain of the adenovirus fiber sequence. Finally, the MEM40 expression cassette comprising the MEM40cDNA, flanked upstream by the CMV promoter and downstream by the bovine growth hormone polyadenylation signal, was inserted into the deleted E3 region.

FIG. 2 schematically shows a construct of Delta-24-MEM40, Delta-24-MEM40 is a replicative adenovirus expressing MEM 40. Delta-24-MEM40 is shown. The construct was similar to DNX-MEM40 (fig. 1) except that it used wild-type adenovirus fibers without insertion of the nucleotide sequence encoding the RGD peptide.

Method of screening for adenoviruses for therapeutic utility

The oncolytic adenoviruses of the present disclosure, or variants or derivatives thereof, can be evaluated for their therapeutic utility by examining their lytic potential in tumor cells. The tumor cells may comprise primary tumor cells derived from a patient biopsy or surgical resection. Alternatively, the tumor cell may comprise a tumor cell line. The cytolytic activity of the adenoviruses of the disclosure can be determined in vitro in tumor cell lines by infecting cells with serial dilutions of the adenovirus and determining the cytolytic potency (e.g., IC 50). Specific methods of determining cytolytic activity may include, but are not limited to, MTS, MTT, and ATP colorimetric assays.

The therapeutic index (the amount of therapeutic agent that causes a therapeutic effect compared to the amount that causes toxicity) of an oncolytic adenovirus of the present disclosure can be calculated by comparing the potency of the cytolytic potency of the adenovirus in a tumor cell line to the cytolytic potency in a matched normal cell.

The oncolytic adenoviruses of the present disclosure can be further evaluated for therapeutic utility by assessing their ability to infect tumor cells and/or normal cells and to express a functional chimeric human/mouse CD40 ligand polypeptide encoded by the oncolytic adenovirus. Chimeric human/mouse CD40 ligand expressed on the cell surface of infected cells can be assessed by flow cytometry using antibodies that specifically recognize human or mouse CD40 antibody binding regions. Chimeric human/mouse CD40 ligand functional activity can also be examined using an in vitro bioassay in which tumor or normal cells infected with an oncolytic adenovirus encoding chimeric human/mouse CD40 ligand are mixed with effector cells that stably express the CD40 receptor fused to a downstream luciferase response element and the induced luciferase expression is measured.

The oncolytic adenoviruses of the present disclosure can be further evaluated for their ability to target tumor cell growth and reduce tumorigenesis or tumor cell burden in mice having naturally derived or transplanted tumors in a mouse's homologous or xenogeneic tumor model. Tumor burden as measured by tumor size, immune protection against tumor re-challenge and animal survival are all possible measures of therapeutic utility and animal tumor models.

Pharmaceutical composition

As described above, there is provided a pharmaceutical composition comprising an adenovirus (as described herein), and one or more pharmaceutically acceptable diluents, carriers or excipients. In some embodiments of the disclosure, the adenovirus is a replication-competent oncolytic adenovirus. In further embodiments, the adenovirus is Delta-24-MEM40 or Delta-24-RGD-MEM 40.

In certain embodiments of the present disclosure, the compositions provided herein may contain buffers, salts, preservatives and other compatible diluents or carriers at pharmaceutically acceptable concentrations. The proportion and nature of the pharmaceutically acceptable diluent is selected to be physiologically compatible and, in particular embodiments, to maintain the viability of the adenovirus. The particular pharmaceutical composition is buffered to an appropriate pH and is isotonic with physiological fluids.

The pharmaceutical compositions provided herein can be prepared by a variety of methods to prepare compositions suitable for administration to a patient such that an effective amount of the active is combined in a mixture with a pharmaceutically acceptable carrier. Suitable carriers are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack publishing Company, Easton, Pa., USA 1985).

The virus solution can be prepared in a physiologically suitable buffer. Representative buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffers is used. The buffering agent or mixture thereof may be present in an amount of about 0.001% to about 10% by weight of the total composition.

Representative procedures and ingredients for selecting and preparing suitable formulations are described, for example, in Remington: the Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1,2005 and The United States PharmacopeE 1A: The National Formulary (USP 40-NF 35and supports).

In various embodiments, the compositions are administered by injection (subcutaneously, intravenously, intramuscularly, etc.) directly at the site of the disease (e.g., tumor site), or by oral administration, or by transdermal administration.

The form of the pharmaceutical composition suitable for injectable use includes sterile aqueous solutions or dispersions (where the term 'sterile' is not intended to indicate that the adenovirus has been killed, indeed, in the particular embodiment described above the adenovirus is replication competent.

In addition, the pharmaceutical compositions may contain preservatives (to the extent that such preservatives do not interfere with the action of the virus.

The adenovirus or pharmaceutical composition comprising the adenovirus may be packaged in a single vial or packaged for administration. Kits containing the virus or pharmaceutical composition can also include instructions for preparing and administering the virus or pharmaceutical composition.

Methods of treatment and administration

In various embodiments of the disclosure, methods for treating cancer are also provided, comprising administering to a subject having a cancer adenovirus as described herein. In further embodiments, the adenovirus is Delta-24-MEM40 or Delta-24-RGD-MEM 40. Regardless of the oncolytic adenovirus and chimeric human/mouse CD40 ligand selected, an oncolytic adenovirus comprising at least one chimeric human/mouse CD40 ligand may be used to treat cancer, whereby the virus is administered by intratumoral injection. However, other routes of delivery are also contemplated, including intravenous, intraperitoneal, intratracheal, intramuscular, intracranial, endoscopic, intralesional, transdermal, subcutaneous, topical, or by direct injection or infusion.

In particular embodiments, the compositions (e.g., pharmaceutical compositions) as described herein are used to treat cancer. As noted above, the term "cancer" as used herein refers to a large family of diseases characterized by uncontrolled growth of cells in vivo. Representative forms of cancer include carcinomas, sarcomas, myelomas, leukemias, lymphomas, and mixed types of the foregoing. Further examples include, but are not limited to, cholangiocarcinoma, bladder cancer, brain gliomas such as glioblastoma, breast cancer, cervical cancer, CNS tumors (such as glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, neuroblastoma, and retinoblastoma), colorectal cancer, endometrial cancer, hematopoietic cell cancers including leukemia and lymphoma, hepatocellular carcinoma, renal cancer, laryngeal cancer, lung cancer, melanoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, squamous cell carcinoma, and thyroid cancer. The cancer may be diffuse (e.g., leukemia), including solid tumors (e.g., sarcomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, and osteogenic sarcoma), or some combination of these (e.g., metastatic cancer with solid tumors and disseminated or diffuse cancer cells). For example, any cancer patient eligible to receive autologous or allogeneic stem cell transplantation will be considered a candidate for this therapy.

In some embodiments, administration can be accomplished by direct administration to the tumor or in situ of the tumor (e.g., after surgical resection or ablation therapy). Administration may be by direct injection or by infusion over a selected period of time. Injection directly into the tumor (intratumoral injection) can be via a thin catheter or cannula. For certain embodiments, the pharmaceutical compositions provided herein can be delivered by a microelectromechanical (MEMS) system under guidance within an MR procedure. In particular, intratumoral injection is achieved without significant reflux or reflux by using a Cannula such as the Alcyone MEMS Cannula (AMC) by Alcyone lifesciences. Representative examples of devices are described in U.S. patent 8,992,458 and U.S. patent publications 2013/0035660, 2013/0035574, and 2013/0035560, each of which is incorporated by reference herein in its entirety.

When administered to a subject, an effective amount of a composition as described herein is administered to treat (e.g., alleviate, ameliorate, relieve, alleviate, stabilize, prevent the spread of, slow or delay the progression of, or cure of) cancer. For example, it may be an amount sufficient to achieve an effect of reducing the number or destroying cancer or tumor cells, or by inhibiting the growth and/or proliferation of such cells. For clinical efficacy, the compositions provided herein can be administered one or more times, depending on the treatment regimen.

Treatment regimens using oncolytic adenoviruses comprising a gene encoding at least one chimeric human/mouse CD40 ligand may include a single administration or multiple administrations. Multiple administrations may be performed over a repeated schedule and/or in response to one or more efficacy indicators of one or more previous administrations or one or more side effects of one or more previous administrations, as will be apparent to one of ordinary skill in the art having the benefit of this disclosure.

The compositions provided herein can be provided in a variety of concentrations. For example, the dose of adenovirus can be provided in a range from a dose greater than about 109 plaque forming units ("pfu"), between about 102 and 109pfu, between about 102 and about 107pfu, between about 103 and about 106pfu, or between about 104 and about 105 pfu.

In some embodiments, the oncolytic adenovirus is administered at a dose of 106-.

The dosage of the pharmaceutical composition to be used may depend on the particular condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size and weight, duration of treatment, nature of concurrent treatment (if any), the particular route of administration, and other similar factors within the knowledge and expertise of a health practitioner. Furthermore, the dosage may depend on the availability of the product.

While not being bound by theory, an oncolytic adenovirus comprising at least one chimeric human/mouse CD40 ligand may have a dual mechanism of action: 1) killing of tumor cells by selective viral replication of oncolytic adenoviruses in cancer cells, 2) induction of systemic anti-tumor immunity resulting from viral immunity induction and immune activation of CD40 ligand.

In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a carrier suitable for administration of the drug to a patient. The pharmaceutically acceptable carrier may vary depending on the route of administration, the storage conditions required for the particular oncolytic adenoviral strain, and other considerations that will be apparent to those of ordinary skill in the art having the benefit of this disclosure. In some embodiments, the pharmaceutically acceptable carrier may be saline.

In some embodiments, the composition may further comprise an adjuvant, such as a viral invasion enhancer of tumor cells, an inducing molecule that induces transcription of a gene in the construct encoding at least one chimeric human/mouse CD40 ligand, wherein the gene is under the control of a promoter activated by an inducer molecule, and the like.

Additional therapies

In some embodiments, the methods may further comprise treatment with an oncolytic adenovirus encoding chimeric human/mouse CD40 in combination with one or more additional therapies. The additional therapy can be radiation therapy, surgery (e.g., lumpectomy or mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, biological therapy, bone marrow transplantation, nano-therapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of an adjuvant or neoadjuvant therapy.

Once a composition is administered to a subject (e.g., a human), the biological activity of the composition can be measured by a variety of methods. Representative parameters that can be evaluated include, for example, imaging, and/or by cytotoxicity assays such as described in, for example, Kochenderfer et al, j.immunotherpy, 32(7): 689-. In certain embodiments, the biological activity of the compositions provided herein can also be measured by determining the expression and/or secretion of certain cytokines (e.g., γ -IFN, IL-2, and TNF). In other embodiments of the present disclosure, biological activity may be assessed by assessing clinical outcome (e.g., reduction in tumor burden or burden).

Any or all of the one or more additional therapies may be administered prior to, concurrently with, or after administration of the oncolytic adenovirus.

Article of manufacture or kit

Also provided herein are articles of manufacture or kits comprising an oncolytic adenovirus encoding a chimeric human/mouse CD40 ligand. The article of manufacture or kit can further comprise a package insert comprising instructions for using the oncolytic adenovirus encoding the chimeric human/mouse CD40 ligand to treat or delay progression of cancer in an individual or to enhance immune function in an individual having cancer. Any oncolytic adenovirus strain encoding a chimeric human/mouse CD40 ligand described herein can be included in an article of manufacture or a kit. Suitable containers include, for example, bottles, vials, bags, and syringes. The container may be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloys (e.g., stainless steel). In some embodiments, the container contains a formulation, and a label on or associated with the container may indicate instructions for use. The article of manufacture or kit may further comprise other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further comprises one or more additional pharmaceutical agents (e.g., chemotherapeutic agents and antineoplastic agents). Suitable containers for one or more medicaments include, for example, bottles, vials, bags, and syringes.

Examples of the invention

Example 1

Construction and characterization of pShuttlE3.2-MEM40

DNA of adenovirus-ISF 35 was isolated using the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer's instructions (batch No. MEM-ADV-FP-009). A1.86 kb PCR fragment containing the cytomegalovirus promoter, the MEM40 coding sequence and the bovine growth hormone polyadenylation sequence was amplified by PCR. The isolated DNA was used as a template and primer pairs containing BglII and MfeI restriction enzyme sites at the 5 'and 3' ends, respectively, were used. The resulting PCR product was digested with BglII and MfeI and ligated into pShutt1E3.2 digested with BamHI and EcoRI, and the MEM40 expression cassette was inserted in the position of E3 in the counterclockwise direction. DH5 cells (Life Technologies) were transformed with a ligation reaction to generate a shuttle vector containing the MEM40 expression cassette (pShutt1E3.2-MEM 40). The pShutt1E3.2-MEM40 clone was sequenced to confirm the integrity of the inserted MEM40 expression cassette.

Example 2

Delta-24-RGD-MEM40 construct

pShutt1E3.2-MEM40 was digested with NaeI to generate a transfer fragment containing the MEM40 cassette and kanamycin resistance gene as well as the left and right flanking regions for recombination. To construct recombinant Delta-24-RGD-MEM40, BJ5138 cells (Agilent Technologies) were co-transformed with the transfer fragment and plasmid pVK526 containing the Delta-24-RGD adenoviral backbone, yielding pVK526-MEM 40/Kan. DH10B cells (Life technologies) were transformed with the obtained pVK526-MEM40/Kan to isolate more concentrated plasmid DNA. pVK526-MEM40/Kan isolated from DH10B cells was subjected to HindIII restriction enzyme digestion and then analyzed on a 1% agarose gel to verify the integrity of the Delta-24-RGD-MEM40 genome. Then, pVK526-MEM40/Kan was digested with SwaI to remove the kanamycin resistance gene and recircularized by ligation to give pVK526-MEM 40.

To rescue Delta-24-RGD-MEM40 virus, pVK526-MEM40 was digested with PacI and transfected into A549 cells using Lipofectamine 3000(Life Technologies) according to the manufacturer's instructions. When cytoplasmic effects were observed, the transfected a549 cell monolayer was harvested and fresh a549 cells were infected with cell lysate to amplify the virus. The resulting virus was verified for the presence of a 24bp deletion in the E1A gene, an RGD insertion in the fiber gene, and an MEM40 expression cassette insertion at the E3 gene position.

Example 3

Characterization of Delta-24-RGD-MEM40

To characterize Delta-24-RGD-MEM40, A549 cells were infected with Delta-24-RGD-MEM40 for 24 hours and analyzed by flow cytometry using PE-labeled antibodies specific for mouse CD154 (clone number MR-1) to confirm cell surface expression of MEM 40. In addition, the biological activity of MEM40 was confirmed using CD40 bioassay kit (Promega Corporation).

The particular embodiments disclosed above are illustrative only, as the disclosure may be modified and practiced in different but equivalent manners apparent to those skilled in the art having the benefit of the teachings herein. For example, the process steps described above may be performed in a different order. Furthermore, no limitations are intended to the details of construction or design herein shown, other than as described in the claims below. It is therefore evident that the particular embodiments disclosed above may be altered or modified and all such variations are considered within the scope and spirit of the disclosure.

Accordingly, the protection sought herein is as set forth in the claims below.

Reference to the literature

The following references, to the extent they provide exemplary procedures or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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aacaaagagg agacgaagaa agatgaggat cctcaaattg cagcacacgt tgtaagcgaa 360

gccaacagta atgcagcatc cgttctacag tgggccaaga aaggatatta taccatgaaa 420

agcaacttgg taaccctgga aaatgggaaa cagctgacgg ttaaaagaca aggactctat 480

tatatctatg ctcaagtcac cttctgctct aatcgggagg cttcgagtca agccccattc 540

atcgtcggcc tctggctgaa gcccagcagt ggatctgaga gaatcttact caaggcggca 600

aatacccaca gttcctccca gctttgcgag cagcagtcta ttcacttggg cggagtgttt 660

gaattacaac caggtgcttc ggtgtttgtc aatgtgactg atccaagcca agtgagccat 720

ggcactggct tcacgtcctt tggcttactc aaactctga 759

<210> 7

<211> 783

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> chimeric human/mouse CD40 ligand (chimeric human/mouse CD40 ligand)

<400> 7

atgatagaaa catacagcca accttccccc agatccgtgg caactggact tccagcgagc 60

atgaagattt ttatgtattt acttactgtt ttccttatca cccaaatgat tggatctgtg 120

ctttttgctg tgtatcttca tagaagattg gataaggtcg aagaggaagt aaaccttcat 180

gaagattttg tattcataaa aaagctaaag agatgcaaca aaggagaagg atctttatcc 240

ttgctgaact gtgaggagat gagaaggcaa tttgaagacc ttgtcaagga tataacgtta 300

aacaaagaag agaaaaaaga aaacagcttt gaaatgcaaa gaggtgatga ggatcctcaa 360

attgcagcac acgttgtaag cgaagccaac agtaatgcag catccgttct acagtgggcc 420

aagaaaggat attataccat gaaaagcaac ttggtaaccc tggaaaatgg gaaacagctg 480

acggttaaaa gacaaggact ctattatatc tatgctcaag tcaccttctg ctctaatcgg 540

gaggcttcga gtcaagcccc attcatcgtc ggcctctggc tgaagcccag cagtggatct 600

gagagaatct tactcaaggc ggcaaatacc cacagttccg ccaagccttg cgggcagcag 660

tctattcact tgggcggagt gtttgaatta caaccaggtg cttcgtgttt tgtcaatgtg 720

actgatccaa gccaagtgag ccatggcact ggcttcacgt cctttggctt actcaaactc 780

tga 783

<210> 8

<211> 759

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> chimeric human/mouse CD40 ligand (chimeric human/mouse CD40 ligand)

<400> 8

atgatcgaaa catacaacca aacttctccc cgatctgcgg ccactggact gcccatcagc 60

atgaaaattt ttatgtattt acttactgtt tttcttatca cccagatgat tgggtcagca 120

ctttttgctg tgtatcttca tagaaggctg gacaagatag aagatgaaag gaatcttcat 180

gaagattttg tattcatgaa aacgatacag agatgcaaca caggagaaag atccttatcc 240

ttactgaact gtgaggagat taaaagccag tttgaaggct ttgtgaagga tataatgtta 300

aacaaagagg agacgaagaa agatgaggat cctcaaattg cagcacacgt tgtaagcgaa 360

gccaacagta atgcagcatc cgttctacag tgggccaaga aaggatatta taccatgaaa 420

agcaacttgg taaccctgga aaatgggaaa cagctgacgg ttaaaagaca aggactctat 480

tatatctatg ctcaagtcac cttctgctct aatcgggagg cttcgagtca agccccattc 540

atcgtcggcc tctggctgaa gcccagcagt ggatctgaga gaatcttact caaggcggca 600

aatacccaca gttccgccaa gccttgcggg cagcagtcta ttcacttggg cggagtgttt 660

gaattacaac caggtgcttc ggtgtttgtc aatgtgactg atccaagcca agtgagccat 720

ggcactggct tcacgtcctt tggcttactc aaactctga 759

<210> 9

<211> 783

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> chimeric human/mouse CD40 ligand (chimeric human/mouse CD40 ligand)

<400> 9

atgatagaaa catacagcca accttccccc agatccgtgg caactggact tccagcgagc 60

atgaagattt ttatgtattt acttactgtt ttccttatca cccaaatgat tggatctgtg 120

ctttttgctg tgtatcttca tagaagattg gataaggtcg aagaggaagt aaaccttcat 180

gaagattttg tattcataaa aaagctaaag agatgcaaca aaggagaagg atctttatcc 240

ttgctgaact gtgaggagat gagaaggcaa tttgaagacc ttgtcaagga tataacgtta 300

aacaaagaag agaaaaaaga aaacagcttt gaaatgcaaa gaggtgatga ggatcctcaa 360

attgcagcac acgttgtaag cgaagccaac agtaatgcag catccgttct acagtgggcc 420

aagaaaggat attataccat gaaaagcaac ttggtaaccc tggaaaatgg gaaacagctg 480

acggttaaaa gacaaggact ctattatatc tatgctcaag tcaccttctg ctctaatcgg 540

gagccttcga gtcaacgccc attcatcgtc ggcctctggc tgaagcccag cagtggatct 600

gagagaatct tactcaaggc ggcaaatacc cacagttcct cccagctttg cgagcagcag 660

tctattcact tgggcggagt gtttgaatta caaccaggtg cttcggtgtt tgtcaatgtg 720

actgatccaa gccaagtgag ccatggcact ggcttcacgt cctttggctt actcaaactc 780

tga 783

<210> 10

<211> 759

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> chimeric human/mouse CD40 ligand (chimeric human/mouse CD40 ligand)

<400> 10

atgatcgaaa catacaacca aacttctccc cgatctgcgg ccactggact gcccatcagc 60

atgaaaattt ttatgtattt acttactgtt tttcttatca cccagatgat tgggtcagca 120

ctttttgctg tgtatcttca tagaaggctg gacaagatag aagatgaaag gaatcttcat 180

gaagattttg tattcatgaa aacgatacag agatgcaaca caggagaaag atccttatcc 240

ttactgaact gtgaggagat taaaagccag tttgaaggct ttgtgaagga tataatgtta 300

aacaaagagg agacgaagaa agatgaggat cctcaaattg cagcacacgt tgtaagcgaa 360

gccaacagta atgcagcatc cgttctacag tgggccaaga aaggatatta taccatgaaa 420

agcaacttgg taaccctgga aaatgggaaa cagctgacgg ttaaaagaca aggactctat 480

tatatctatg ctcaagtcac cttctgctct aatcgggagc cttcgagtca acgcccattc 540

atcgtcggcc tctggctgaa gcccagcagt ggatctgaga gaatcttact caaggcggca 600

aatacccaca gttcctccca gctttgcgag cagcagtcta ttcacttggg cggagtgttt 660

gaattacaac caggtgcttc ggtgtttgtc aatgtgactg atccaagcca agtgagccat 720

ggcactggct tcacgtcctt tggcttactc aaactctga 759

<210> 11

<211> 783

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> chimeric human/mouse CD40 ligand (chimeric human/mouse CD40 ligand)

<400> 11

atgatagaaa catacagcca accttccccc agatccgtgg caactggact tccagcgagc 60

atgaagattt ttatgtattt acttactgtt ttccttatca cccaaatgat tggatctgtg 120

ctttttgctg tgtatcttca tagaagattg gataaggtcg aagaggaagt aaaccttcat 180

gaagattttg tattcataaa aaagctaaag agatgcaaca aaggagaagg atctttatcc 240

ttgctgaact gtgaggagat gagaaggcaa tttgaagacc ttgtcaagga tataacgtta 300

aacaaagaag agaaaaaaga aaacagcttt gaaatgcaaa gaggtgatga ggatcctcaa 360

attgcagcac acgttgtaag cgaagccaac agtaatgcag catccgttct acagtgggcc 420

aagaaaggat attataccat gaaaagcaac ttggtaaccc tggaaaatgg gaaacagctg 480

acggttaaaa gacaaggact ctattatatc tatgctcaag tcaccttctg ctctaatcgg 540

gagccttcga gtcaacgccc attcatcgtc ggcctctggc tgaagcccag cagtggatct 600

gagagaatct tactcaaggc ggcaaatacc cacagttccg ccaagccttg cgggcagcag 660

tctattcact tgggcggagt gtttgaatta caaccaggtg cttcggtgtt tgtcaatgtg 720

actgatccaa gccaagtgag ccatggcact ggcttcacgt cctttggctt actcaaactc 780

tga 783

<210> 12

<211> 759

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> chimeric human/mouse CD40 ligand (chimeric human/mouse CD40 ligand)

<400> 12

atgatcgaaa catacaacca aacttctccc cgatctgcgg ccactggact gcccatcagc 60

atgaaaattt ttatgtattt acttactgtt tttcttatca cccagatgat tgggtcagca 120

ctttttgctg tgtatcttca tagaaggctg gacaagatag aagatgaaag gaatcttcat 180

gaagattttg tattcatgaa aacgatacag agatgcaaca caggagaaag atccttatcc 240

ttactgaact gtgaggagat taaaagccag tttgaaggct ttgtgaagga tataatgtta 300

aacaaagagg agacgaagaa agatgaggat cctcaaattg cagcacacgt tgtaagcgaa 360

gccaacagta atgcagcatc cgttctacag tgggccaaga aaggatatta taccatgaaa 420

agcaacttgg taaccctgga aaatgggaaa cagctgacgg ttaaaagaca aggactctat 480

tatatctatg ctcaagtcac cttctgctct aatcgggagc cttcgagtca acgcccattc 540

atcgtcggcc tctggctgaa gcccagcagt ggatctgaga gaatcttact caaggcggca 600

aatacccaca gttccgccaa gccttgcggg cagcagtcta ttcacttggg cggagtgttt 660

gaattacaac caggtgcttc ggtgtttgtc aatgtgactg atccaagcca agtgagccat 720

ggcactggct tcacgtcctt tggcttactc aaactctga 759

Claims (38)

1. A composition, comprising:

a replicative oncolytic adenovirus comprising a heterologous nucleic acid inserted into a non-essential region of the adenovirus genome, said nucleic acid comprising a sequence encoding a CD40 agonist operably linked to a transcriptional control element.

2. The composition of claim 1, wherein the replication competent oncolytic adenovirus comprises a deletion of part or all of the E3 gene region.

3. The composition of claim 2, wherein a nucleic acid comprising a sequence encoding a CD40 agonist operably linked to a transcriptional control element is inserted into the gene region of E3.

4. The composition of claim 3, wherein the nucleic acid comprising a sequence encoding a CD40 agonist operably linked to a transcriptional control element is inserted in an opposite orientation to a native E3 gene.

5. The composition of claim 1, wherein the CD40 agonist is at least one CD40 ligand (CD 40L).

6. The composition of claim 5, wherein at least one CD40L is a chimeric human/mouse CD40 ligand.

7. The composition of claim 6, wherein at least one chimeric human/mouse CD40 ligand is selected from the group consisting of ISF30(SEQ ID NO: 1), ISF31(SEQ ID NO: 2), ISF32(SEQ ID NO: 3), ISF33(SEQ ID NO: 4), ISF34(SEQ ID NO: 5), ISF35(MEM40) (SEQ ID NO: 6), ISF36(SEQ ID NO: 7), ISF37(SEQ ID NO: 8), ISF38(SEQ ID NO: 9), ISF39(SEQ ID NO: 10), ISF40(SEQ ID NO: 11), and ISF41(SEQ ID NO: 12).

8. The composition of claim 7, wherein at least one chimeric human/mouse CD40 ligand is MEM 40.

9. The composition of claim 6, wherein at least one chimeric human/mouse CD40 ligand has at least 90% identity to a ligand selected from the group consisting of ISF30(SEQ ID NO: 1), ISF31(SEQ ID NO: 2), ISF32(SEQ ID NO: 3), ISF33(SEQ ID NO: 4), ISF34(SEQ ID NO: 5), ISF35(MEM40) (SEQ ID NO: 6), ISF36(SEQ ID NO: 7), ISF37(SEQ ID NO: 8), ISF38(SEQ ID NO: 9), ISF39(SEQ ID NO: 10), ISF40(SEQ ID NO: 11), and ISF41(SEQ ID NO: 12).

10. The composition of claim 1, wherein the transcriptional control element operably linked to the CD40 agonist is a transcriptional promoter.

11. The composition of claim 10, wherein the transcription promoter is a Cytomegalovirus (CMV) promoter.

12. The composition of claim 1, wherein the replication competent oncolytic adenovirus is a human type 5 adenovirus.

13. The composition of claim 12, wherein the replication competent oncolytic adenovirus is a Delta 24 adenovirus or a Delta-24-RGD adenovirus.

14. The composition of claim 1, further comprising a pharmaceutically acceptable carrier.

15. A method, comprising:

administering to a patient having cancer a replication competent oncolytic adenovirus having a heterologous nucleic acid inserted into a non-essential region of the adenovirus genome, said nucleic acid comprising a sequence encoding a CD40 agonist operably linked to a transcriptional control element.

16. The method of claim 15, wherein the replication competent oncolytic adenovirus comprises a deletion of part or all of the E3 gene region.

17. The method of claim 16, wherein a nucleic acid comprising a sequence encoding a CD40 agonist operably linked to a transcriptional control element is inserted into the gene region of deletion E3.

18. The method of claim 17, wherein the nucleic acid comprising a sequence encoding a CD40 agonist operably linked to a transcriptional control element is inserted in an opposite orientation to a native E3 gene.

19. The method of claim 15, wherein the replication competent oncolytic adenovirus is a Delta 24 adenovirus or a Delta-24-RGD adenovirus.

20. The method of claim 15, wherein the CD40 agonist is at least one CD40 ligand (CD 40L).

21. The method of claim 15, wherein at least one CD40L is a chimeric human/mouse CD40 ligand.

22. The method of claim 21, wherein at least one chimeric human/mouse CD40 ligand is selected from the group consisting of ISF30(SEQ ID NO: 1), ISF31(SEQ ID NO: 2), ISF32(SEQ ID NO: 3), ISF33(SEQ ID NO: 4), ISF34(SEQ ID NO: 5), ISF35(MEM40) (SEQ ID NO: 6), ISF36(SEQ ID NO: 7), ISF37(SEQ ID NO: 8), ISF38(SEQ ID NO: 9), ISF39(SEQ ID NO: 10), ISF40(SEQ ID NO: 11), and ISF41(SEQ ID NO: 12).

23. The method of claim 22, wherein at least one chimeric human/mouse CD40 ligand is MEM 40.

24. The method of claim 21, wherein at least one chimeric human/mouse CD40 ligand has at least 90% identity to a ligand selected from the group consisting of ISF30(SEQ ID NO: 1), ISF31(SEQ ID NO: 2), ISF32(SEQ ID NO: 3), ISF33(SEQ ID NO: 4), ISF34(SEQ ID NO: 5), ISF35(MEM40) (SEQ ID NO: 6), ISF36(SEQ ID NO: 7), ISF37(SEQ ID NO: 8), ISF38(SEQ ID NO: 9), ISF39(SEQ ID NO: 10), ISF40(SEQ ID NO: 11), and ISF41(SEQ ID NO: 12).

25. The method of claim 15, wherein the transcriptional control element operably linked to the CD40 agonist is a transcriptional promoter.

26. The method of claim 25, wherein the transcription promoter is a Cytomegalovirus (CMV) promoter.

27. The method of claim 15, wherein the oncolytic adenovirus is administered as a composition comprising a pharmaceutically acceptable carrier.

28. The method of claim 15, wherein the patient has a cancer selected from primary or metastatic cancer.

29. The method of claim 15, wherein the patient has brain or bladder cancer.

30. The method of claim 15, wherein the oncolytic adenovirus is administered intratumorally, intravenously, intraperitoneally, intratracheally, intramuscularly, intracranially, endoscopically, intralesionally, transdermally, subcutaneously, regionally or by direct injection or infusion.

31. The method of claim 15, wherein the oncolytic adenovirus is administered one or more times.

32. The method of claim 31, wherein the replication competent oncolytic adenovirus is present at 10⁶-10¹³The dose of plaque forming units (pfu) was administered.

33. The method of claim 15, further comprising:

administering at least one additional therapeutic agent.

34. The method of claim 33, wherein at least one additional therapeutic agent is chemotherapy, immunotherapy, surgery, radiation therapy, viral therapy, or biological therapy.

35. The method of claim 34, wherein the at least one additional therapeutic agent is administered to the patient prior to administration of the replication competent oncolytic adenovirus.

36. The method of claim 34, wherein the at least one additional therapeutic agent is administered to the patient concurrently with the administration of the replication competent oncolytic adenovirus.

37. The method of claim 34, wherein the at least one additional therapeutic agent is administered to the patient after administration of the replication competent oncolytic adenovirus.

38. The method of claim 15, wherein the patient is a human.

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